The long-term objective of the proposed research is to understand the molecular mechanisms of Wnt4 signaling in mammalian female sex determination. Wnt4 is associated with partial sex reversal. Male patients with duplications in Wnt4 exhibit sexual phenotypes ranging from cryptorchidism to XY sex reversal. A female patient carrying a Wnt4 mutation lacks structures of the female reproductive tract. Clearly, defining Wnt4 action would not only add to our basic understanding of sex determination, but would also improve management of human sexual pathologies. In the field to date, only two possible Wnt4 target genes have been identified, and they are not fully characterized. Specific Aim 1 tests the hypothesis that Wnt4 acts through a novel signaling pathway based on in vitro data showing the relocalization of beta-catenin to the cytoplasmic membrane after Wnt4 stimulation. We will identify the detailed expression profile of Wnt4 in the developing ovary, using in situ hybridization and immunohistochemistry (IHC) with newly available cell markers; we will examine the localization of beta-catenin-the proposed mediator of Wnt4 function-in wild type and Wnt4 mutant embryos; finally we will assay the phosphorylation state of Beta-catenin by Western blot analysis. Specific Aim 2 tests the hypotheses that Wnt4 action regulates two genes, angiomotin and caveolin-1, which are both involved in processes seen in the Wnt4 phenotype: cell migration and angiogenesis. This aim will be accomplished using Wnt4-transfected cells to examine target gene expression, cell aggregation, migration and tubule formation; by assessing target gene expression in Wnt4 wild type and mutant embryos using IHC; and by examining ttie phenotype of caveolin-1 null gonads during development.